The comprehensive analysis of low-abundance analytes in ultrasmall volumes of complex biological matrices—including samples from single cells—is one of the main challenges of today's science and technology. To address this challenge, efficient concentration and separation techniques are critically needed.
Concentration polarization is a well known phenomenon in which part of a liquid is depleted of ions. For the transport and separation of charged molecules in capillaries and microchannels, electrophoresis is often employed and has found widespread applications. However, the sensitivity and selectivity to accurately handle and separate substances in tubing is limited, thus requiring efficient sample preconcentration methods.
For ionic analytes, isotachophoresis (ITP) has proven as a powerful candidate, as disclosed by B. Jung et al., Analytical Chemistry, vol. 78, pp. 2319-2327, (2006); R. B. Schoch et al., Lab on a Chip, vol. 9, pp. 2145-2152, (2009).
ITP uses an imposed electrophoretic mobility gradient to create concentrated analyte zones with non-dispersing interfaces in an elongated channel. Analyte ions to be stacked and separated are typically introduced between a leading and a trailing electrolyte with an effective mobility respectively higher and lower than those of the analytes. Under the influence of an electric field, analyte ions redistribute themselves into sequential zones in order of reducing effective mobility, starting from the leading, and ending with the trailing electrolyte. ITP based separations typically result in adjacent, contiguous zones of analytes moving at identical speed downstream in a main separation channel.
Use of isotachophoresis for separation and detection of charged components typically requires additional steps, typically also involving a secondary separation method. Examples include pre-concentration of compounds by isotachophoresis followed by electrophoresis and analysis, as disclosed in US-A-2006/0254915, US-A-2002/0189946; isotachophoresis followed by zone electrophoretic separation.
Transient isotachophoresis (tITP) couples the concentration characteristics of isotachophoresis with the resolving power of zone electrophoresis. However, tITP is of significant complexity, as it requires injection of three electrolyte zones (background electrolyte, sample, background electrolyte) and the moment of transience is difficult to monitor.
Furthermore, to be effective, tITP may require the use of a electrolytes with specific chemical and physical properties, as exemplified in U.S. Pat. No. 5,817,225 and WO-A-2009/079028.
Accordingly, there remains a need to separate multiple compounds in complex samples, without the need to operate with different electrolytes, and with the potential to not only detect and analyse components, but also to selectively separate the components.